Posttranslational histone modifications play important roles in regulating chromatin structure and function. One example of histone modification is ubiquitination, which occurs predominately on H2A and H2B. Although recent studies have revealed a critical role of H2A ubiquitination in Hox gene silencing and X inactivation, the function of H2A deubiquitination is unknown and the enzymes that catalyze H2A deubiquitination have not been identified. In the preliminary results presented in this proposal, we identified Ubp-M as the deubiquitinase for histone H2A. Further characterization revealed that Ubp-M-mediated H2A deubiquitination plays important roles in cell cycle progression and gene expression. In this proposal we propose to thoroughly characterize Ubp-M, with the long-range goal of determining the roles and elucidating the mechanisms of Ubp-M and its mediated H2A deubiquitination in chromatin and cellular regulation. Our hypothesis is that Ubp-M regulates cell cycle progression and gene expression, in part, through H2A deubiquitination. To address this hypothesis, we have established three Specific Aims to: 1. Characterize the functional domains of Ubp-M by defining regions in Ubp-M that are necessary for its nucleosomal- and H2A-specific deubiquitination in vitro and in vivo as well as for its normal subcellular localization. 2. Determine the role of phosphorylation in regulating Ubp-M's function by mapping the phosphorylation sites and investigating how mutations of these sites regulate Ubp-M's function in vitro and in vivo. Furthermore, the roles of phosphorylation in regulating Ubp-M's nuclear localization signal (NLS) and nuclear export signal (NES) will be investigated. 3. Identify and characterize Ubp-M interacting proteins by first confirming their interactions with Ubp-M and mapping the sites of protein-protein interaction, and then determining how these interacting proteins regulate Ubp-M's cellular functions in vitro and in vivo. In addition to these three Specific Aims, we outline plans to generate Ubp-M complete and conditional knockout mice and describe our experimental progress. Our preliminary studies indicate that Ubp-M regulates early embryogenesis in Xenopus laevis and is highly expressed in ovarian and breast cancer cells, underscoring the importance to determine the function of Ubp-M in vivo. However, due to the amount of work in this application and the uncertain outcome of these experiments, we did not list these experiments as a Specific Aim.